Bacterial virulence genes

ABSTRACT

An attenuated  Actinobacillus pleuropneumoniae  bacterium has a mutation in a gene required for bacterial virulence. Vaccines based upon the bacterium are provided, as are isolated virulence genes and polypeptides and uses thereof.

The present invention relates to the identification of genes involved inthe virulence of Actinobacillus pleuropneumoniae, to the production ofattenuated mutant strains of Actinobacillus pleuropneumoniae, and to theidentification of anti-bacterial agents which target the identifiedgenes and their products. A. pleuropneumoniae is the causative agent ofporcine pleuropneumonia, a highly contagious, respiratory disease ofpigs. The disease is often fatal and as a result leads to severeeconomic loss in the swine producing industry. An obligate parasite ofthe porcine respiratory tract, the incidence of infection of A.pleuropneumoniae has increased in recent years, notably where animalsare subjected to intensive breeding conditions. Transmission of thebacterium is by aerosol, or by direct contact with infected pigs.

Pleuropneumonia can occur in swine of all ages, and there are no knownassociations with predisposing viral or bacterial infections. A.pleuropneumoniae infection may be chronic or acute, with the diseasebeing characterised by increasingly severe pulmonary distress, whichprogresses rapidly to death, or to a chronic infection resulting in afailure to thrive. A hemorrhagic, necrotic bronchopneumonia withaccompanying fibrinous pleuritis is typically observed.

Fifteen different serotypes of A. pleuropneumoniae have been identifiedbased on antigenic differences in their capsular polysaccharides, andtheir production of extracellular toxins. Serotypes 1, 5 and 7 are themost common forms of A. pleuropneumoniae infection in the United States,whereas serotypes 1, 2, 5, 7 and 9 predominate in Europe.

Treatment of pigs infected with A. pleuropneumoniae usually involves thedirect injection of an antibiotic (typically tetracycline). Such amethod is however labour intensive, time consuming, and expensive, andcan often be of limited use due to the rapid progression of the disease.Evidence from field and experimental studies has however shown thatinfection with A. pleuropneumoniae can provide a lifetime protectionagainst subsequent reinfection, thereby suggesting that vaccinationmight be a feasible alternative to antibiotic therapy. The developmentof an effective vaccine has so far been hampered by the antigenicdiversity of the fifteen different serotypes.

In attempts to produce a vaccine composition, killed, whole cellbacteria were found to provide only serotype-specific protection, but ithas been shown that natural infection with a highly virulent serotypecan stimulate a strong protective cross-immunity against multipleserotypes [Nielsen, Nord Vet Med. 31: 407-13 (1979); Nielsen, Nord VetMed. 36: 221-234 (1984); Nielsen, Can J Vet Res. 29: 580-582 (1988);Nielsen, ACTA Vet Scand. 15: 80-89 (1994)]. Certain undefined liveattenuated mutants have also shown promise for cross-protection of swine[Inzana et al, Infect Immun. 61: 1682-6 (1993); Paltineanu et al, InInternational Pig Veterinary Society, 1992, p. 214; Utrera et al, InInternational Pig Veterinary Society, 1992, p. 213] but because of theuncertainties associated with vaccines comprising bacterial strainshaving such undefined spontaneous mutations, there exists a need in theart for the rational construction of live attenuated strains which willsafely stimulate protective immunity against homologous, and preferablyheterologous Actinobacillus pleuropneumoniae serotypes. There alsoexists a need to identify the genes involved in A. pleuropneumoniaevirulence, to facilitate the development of methods for identifyinganti-bacterial agents which target those genes and/or their products.

Published International patent application WO 00/61724 (Pharmacia &Upjohn) describes the use of signature tagged mutagenesis (STM) toidentify genes responsible for the virulence of gram-negative bacteria.The development of vaccines containing attenuated bacteria is alsodiscussed. The work is also described in Fuller et al., MicrobialPathogenesis [2000] vol. 26; pp. 39-51.

In the STM technique, a plurality of different bacterial strains, eachof which carries a random mutation in its genome, is produced by meansof transposon integration. The inserted transposons carry different DNAsignature tags, which allows individual mutants to be differentiatedfrom one other. The tags each comprise a variable central region flankedby invariant “arms”, the latter of which allow the variable centralportion to be amplified by PCR. Multiple tagged mutant strains arecollected in microtiter dishes, and then combined to form an “inoculumpool” for animal infection.

At an appropriate time after infection, bacteria are isolated from theinfected animal, and are combined to form a “recovered pool.” The tagsin the recovered pool and the tags in the inoculum pool are separatelyamplified, labelled, and used to probe filters arrayed with all thedifferent tags from the mutants in the inoculum. Mutant strains withattenuated virulence are generally those which cannot be recovered fromthe infected animal, i.e. strains with tags that give hybridizationsignals when probed with tags from the inoculum pool, but not whenprobed with tags from the recovered pool. The advantage of STM is that alarge number of insertional mutant strains can be simultaneouslyscreened in a single animal for loss of bacterial virulence. STM is morefully described in e.g. WO 96/17951.

After considerable research, the present inventors have providedimproved methods and means for identifying genes involved in A.pleuropneumoniae virulence. This has led to the identification of a newfamily of “virulence genes” the members of which each comprise anucleotide sequence which is selected from any one of those defined bySEQ ID NO: 1-56. As used herein, the term “virulence genes” refers togenes of a bacterium whose correct function is involved in, e.g.required for the establishment and/or maintenance of an infection in ahost animal. Virulence genes and their products, which may bepolynucleotides (e.g. rRNA) and/or polypeptides, are thus involved inthe pathogenesis of the bacterium, but might not be necessary for itsgrowth in vitro.

Mutations in virulence genes can result in an “attenuated bacterium”,i.e. one whose ability to establish and/or maintain a bacterialinfection in a host animal is reduced, as compared to a correspondingwild-type bacterium of the same strain and serotype. Attenuation of thebacterium may be confirmed by means of its administration to a subjectanimal, where its inability to cause a sustained infection leading toe.g. chronic infection or death may be observed. The non-establishmentof symptoms of porcine pleuropneumonia, e.g. of pulmonary distress,hemorrhagic necrotic bronchopneumonia and/or fibrinous pleuritis, mayalso be used as an index of bacterial attenuation. Attenuated bacteriaare of commercial importance in vaccine compositions.

In one aspect, the present invention thus provides an attenuatedActinobacillus pleuropneumoniae bacterium.

In a particular embodiment, the present invention provides an attenuatedActinobacillus pleuropneumoniae bacterium having a mutation in a genewhich comprises a nucleotide sequence selected from the group consistingof SEQ ID NO.:1-56.

The mutation may inhibit or prevent the function of the gene, e.g. byreducing the level of its expression (e.g. by reducing transcriptionand/or translation), and/or by impairing the biological activity of thegene product, e.g. by causing an inactive form of the gene product thatis encoded by the mutated gene to be produced. The gene product may be apolynucleotide or polypeptide.

The mutation in the gene may be an insertion, deletion or substitution.It may be located in a region of the gene that encodes a polypeptide, orin a sequence responsible for, or involved in, the control of geneexpression, e.g. in a promoter region. Mutations which involve acombination of one or more of insertion, deletion and/or substitutionare also contemplated. Attenuated A. pleuropneumoniae strains of thepresent invention likewise include those which have two or moremutations, each of which may be an insertion, a deletion, asubstitution, or a combination thereof. Multiple mutations may occur ina single gene, or they may occur in different genes within the samebacterial genome. They may result in an additive or synergistic level ofattenuation. Multiple mutations may be prepared by design, or mayfortuitously arise as a result of techniques for introducing a singlemutation.

Deletion mutants include those in which all or part of the virulencegene is deleted. In one aspect, the mutation results in the deletion ofat least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 98%, or at least about 99% of the virulence gene. A deletion mayoccur in the appropriate nucleotide sequence set forth in SEQ IDNO.:1-56, or it may occur in an adjacent region of the gene, e.g. in anassociated control sequence.

Deletion mutants can be constructed using any technique which is wellknown to those skilled in the art. One strategy employscounter-selectable markers, and has been used to delete genes in manydifferent bacteria. For details of this technique, reference may be madeto e.g. Reyrat et al, Infection and Immunity [1998] vol. 66; pp.4011-4017, and Oswald et al., FEMS Microbiol Lett [1999] vol. 179; pp.153-160.

The process employs a double selection strategy, in which a plasmid isconstructed which encodes both a selectable and a counter-selectablemarker, with flanking DNA sequences being derived from both sides of thedesired deletion site in the bacterial genome. The selectable marker isused to select for bacteria in which the plasmid has integrated into thegenome in the appropriate location and manner. The counter-selectablemarker is then used to select for that small percentage of bacteriawhich spontaneously eliminate the integrated plasmid. A fraction ofthose resultant bacteria will contain the desired deletion, with noforeign DNA remaining.

In another technique for achieving a deletion, the cre-lox system isused for site specific recombination of DNA. The system consists of 34base pair lox sequences that are recognised by a bacterial crerecombinase. If the lox sites are introduced into the genomic DNA in anappropriate orientation, a sequence which is flanked by the lox sitescan be excised by expression of the cre recombinase, one copy of the loxsite being retained. Using standard recombination techniques, it ispossible to delete a sequence of interest in the A. pleuropneumoniaegenome and to replace it with a selectable marker (e.g. a gene encodingkanamycin resistance) which is flanked by lox sites. Transientexpression of the cre recombinase (e.g. from an electroporated suicide(non-replicating) plasmid containing the cre gene under the control of apromoter which functions in A. pleuropneumoniae) will then result inefficient elimination of the lox flanked marker. This process results ina bacterial mutant containing the desired deletion, and one copy of thelox sequence.

In another approach to providing a deletion, a target sequence in the A.pleuropneumoniae genome can be deleted (replaced) with a marker gene,e.g. a green fluorescent protein (GFP), β-galactosidase or luciferase.DNA segments flanking a desired deletion are prepared by PCR and clonedinto a suicide vector for A. pleuropneumoniae. An expression cassette,containing a promoter active in A. pleuropneumoniae and operably linkedto the appropriate marker gene, is then cloned between the flankingsequences. The plasmid is introduced into wild-type bacteria and mutantswhich both incorporate and express the marker gene are isolated andexamined for a desired recombination event (replacement of the wild-typegene with the marker gene).

As for insertion mutants, these include genes in which a stop codon isinserted into an open reading frame (ORF) upstream (5′) of the native,i.e. endogenous stop signal. A truncated, inactive form of the(polypeptide) virulence gene product may thus be expressed by the gene.Insertions which cause a frame-shift are likewise contemplated for theproduction of inactive products. Mutated virulence genes of A.pleuropneumoniae will also include those in which a transposon has beeninserted into a protein-coding or regulatory sequence of the gene.Attenuated bacteria produced by the STM technique are insertionalmutants in which a virulence gene has been rendered non-functional byinsertion of a transposon sequence, e.g. Tn10.

Because insertional mutants, e.g. those generated by STM, will stillcontain all of the genetic information required for bacterial virulence,there is a risk that they could revert to a pathogenic state, e.g. byspontaneous deletion of the inserted sequence. Accordingly, whenpreparing a vaccine or other therapeutic composition, based on aninsertional mutant, e.g one identified by STM, it may be desirable totake the information obtained from that STM-derived strain, i.e. theidentification of the disrupted virulence gene, and then recreate amutant in which some, most, or all of that virulence gene has insteadbeen deleted. This should preclude the possibility that the bacteriumwill revert to a virulent state, thereby to cause pathology, rather thanprophylactic protection of a host animal to which it is administered.

In a related aspect, the present invention provides a compositioncontaining the attenuated A. pleuropneumoniae bacterium according to thefirst aspect of the invention. The composition may be an immunogeniccomposition, i.e. one which is capable of eliciting an antibody responsein a host animal to which it is administered. The composition may be atherapeutic (e.g. prophylactic) composition. It may be a vaccine,preferably one whose administration to an animal confers a degree ofprotection against subsequent infection with serotypes and strains of A.pleuropneumoniae different from that/those which is/are present in thevaccine itself (cross-protection). The invention further provides forthe use of an attenuated A. pleuropneumoniae bacterium of the firstaspect of the invention for the manufacture of a medicament (e.g.vaccine) for preventing or alleviating an infection of an animal with A.pleuropneumoniae and/or for preventing or alleviating symptomsassociated with such infection, e.g. for the prophylactic protection ofswine against porcine pleuropneumonia.

In order for an attenuated bacterium to be effective in a vaccinecomposition, the (one or more) mutation(s) in the virulence gene(s)should be significant enough to prevent the pathogen from evoking severeclinical symptoms, but insignificant enough to allow a limitedreplication and growth of the bacteria in the host animal, thereby toelicit appropriate defence mechanisms resulting in immunological memory.Mutations in genes which are required for (as distinct from merelyinvolved in) virulence are preferred.

Compositions of the invention may contain a plurality of differentattenuated A. pleuropneumoniae bacteria, e.g. bacteria having differentmutations in the same virulence gene, and/or bacteria having similar, ordifferent, mutations in two or more different genes. While it ispossible for an avirulent (attenuated) bacterium of the presentinvention to be administered alone, it is preferably administered in asolid or liquid composition containing one or more suitable adjuvants,diluents, carriers, vehicles and/or excipients, as discussed elsewhereherein. Compositions according to, and for use in the present invention,may thus comprise one or more of these substances, which should bepharmacologically “acceptable”, in the sense of being both compatiblewith the attenuated bacterium, and not deleterious to the animal to beimmunized. Additional substances are preferably both sterile and pyrogenfree.

Examples of adjuvants which may be used in the present invention includeoil-based adjuvants such as Freund's Complete Adjuvant and Freund'sIncomplete Adjuvant, mycolate-based adjuvants (e.g. trehalosedimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (e.g.mureins, mucopeptides, or glycoproteins such as N-Opaca, muramyldipeptide [MDP], or MDP analogs), proteoglycans (e.g. those extractedfrom Klebsiella pneumoniae), streptococcal preparations (e.g. OK432),Biostim™ (e.g. O1K2), the “Iscoms” of EP 109942, EP 180 564 and EP 231039, aluminum hydroxide, saponin, DEAE-dextran, neutral oils (such asmiglyol), vegetable oils (such as arachis oil), liposomes, Pluronicpolyols, the Ribi adjuvant system (see e.g. GB-A-2 189 141) andinterleukins, particularly those that stimulate cell-mediated immunity.An adjuvant consisting of extracts of Amycolata, a genus of bacteria ofthe order Actinomycetales, has also been described (U.S. Pat. No.4,877,612). Proprietary adjuvant mixtures are commercially available.The adjuvant to be used will depend, at least in part, on the recipientanimal. The amount of adjuvant to be administered will also depend onthe type and size of the animal. Optimal dosages may be readilydetermined by routine methods well known to those skilled in the art.

As for any diluent(s), carrier(s), vehicle(s) and/or excipient(s), thesemay be liquid, semisolid, or solid in nature. Any suitable substanceknown to those skilled in the art may be used. Exemplary substancesinclude polyoxyethylene sorbitan monolaurate, magnesium stearate, methyland propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose,dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineraloil, cocoa butter, and oil of theobroma.

The compositions of the invention may be packaged in various forms whichmaybe convenient for, or adapted to, a particular route of delivery.Therapeutic compositions (e.g. vaccines) may be suitable foradministration to a subject animal by for example intravenous,intradermal, intramuscular, intramammary, intraperitoneal and/orsubcutaneous injection, and/or by oral, sublingual, nasal, anal orvaginal delivery. The treatment may consist of a single dose, or of aplurality of doses over a period of time. Decisions on administrationregimes are well within the ambit of those skilled in the art. Thecomposition may be formulated and packaged as appropriate.

In a further aspect, the present invention provides an isolatedpolynucleotide encoding a gene product which is naturally involved in(e.g. required for) the virulence of A. pleuropneumoniae. The sequenceof the polynucleotide is thus found in a virulence gene of A.pleuropneumoniae. The invention also provides for a polynucleotideencoding a gene product which is not naturally found in A.pleuropneumoniae, but whose expression therein is capable of modulating(e.g. of decreasing) the virulence of that bacterium. Such apolynucleotide may be introduced into the bacterium to cause suchexpression (e.g. by transformation, transfection or electroporation).Expression may result in an attenuated strain of the bacterium, e.g. foruse in a vaccine or other immunogenic composition. Polynucleotides whichare not naturally found in A. pleuropneumoniae but which are capable ofmodulating the virulence of that bacterium by their direct interactionwith A. pleuropneumoniae virulence genes or gene products (rather thanby indirect action through their own gene products) are alsocontemplated.

Any gene product of a polynucleotide according to the present inventionmay be a polynucleotide (e.g. RNA) or a polypeptide. Polypeptide geneproducts are herein referred to as “virulence polypeptides”, and areinvolved in/capable of modulating the virulence of A. pleuropneumoniae,whether or not they are naturally encoded by the wild-type bacterialgenome. Polynucleotide gene products which can affect the virulence ofA. pleuropneumoniae include e.g. antisense RNA and ribozymes. An effecton bacterial virulence may be demonstrated for any gene product bydetermining the extent to which a strain of the bacterium A.pleuropneumoniae which expresses the product can establish and/ormaintain an infection in an animal as compared with A. pleuropneumoniaeof the same strain and serotype which does not express the product.

According to this aspect, the present invention more particularlyprovides for an isolated polynucleotide comprising (e.g. consisting of):(a) a nucleotide sequence selected from the group consisting of SEQ IDNO.: 1-56 (such sequences are found within the virulence genes of A.pleuropneumoniae as identified elsewhere herein);

(b) a nucleotide sequence encoding the polypeptide which is encoded bythe nucleotide sequence recited in (a); (c) a nucleotide sequence whichhybridizes to the nucleotide sequence of (a) and/or (b), or to itscomplement, under conditions of moderate to high stringency; or (d) afragment of any one of the nucleotide sequences of (a)-(c), whichfragment retains an immunological property and/or a biological activityof the recited nucleotide sequence of (a)-(c). Polynucleotides of thepresent invention may be DNA or RNA, as described elsewhere herein.

Nucleotide sequences of type (b) can be readily identified once an aminoacid sequence is derived from the nucleotide sequence of (a), andaccount is taken of the genetic code.

As to (c), exemplary conditions of moderate stringency include a finalwash in a buffer comprising 2×SSC/0.1% SDS, at 35° C. to 45° C.Exemplary conditions of high stringency include a final wash in a buffercomprising 0.2×SSC/0.1% SDS, at 65° C. to 75° C. It is understood in theart that conditions of equivalent stringency can be achieved by means ofvariation of temperature and buffer or of salt concentration. In thisregard, reference may be made to Ausubel et al., Protocols in MolecularBiology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications inhybridization conditions can be empirically determined or preciselycalculated based on length and percentage of guanosine/cytosine (GC)base pairing. Hybridization conditions can be calculated as described inSambrook et al, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to9.51.

For fragments of type (d), knowledge of the nucleotide sequenceaccording to (a), (b) or (c) makes available to the skilled person everypossible fragment of that nucleotide sequence. Fragments may be at least10, at least 15, at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, or at least 50 nucleotides in length. They may becapable of hybridising to the nucleotide sequence of (a), (b) or (c), orto its complement, under conditions of moderate to high stringency,optionally in the presence of competitive nucleic acid, e.g. in thepresence of nucleic acid from a cDNA or mRNA library derived from A.pleuropneumoniae. The retained immunological property may be an abilityof the fragment to be bound specifically by an antibody which bindsspecifically to the full length nucleotide sequence. Binding affinitiesof at least 10⁷ M⁻¹ are contemplated. A retained biological activity maybe an ability of the fragment to encode a polypeptide which shares abinding capability with the polypeptide encoded by the full lengthnucleotide sequence, e.g an ability to bind to one of the ligands towhich it naturally binds in vivo, or the same antibody.

As will be readily apparent to the person of skill in the art, theidentification of virulence genes of Actinobacillus pleuropneumoniaeenables the identification of corresponding (i.e. homologous) genes inother related bacteria, e.g. in other members of the Pasteurellaceaefamily. In isolated form, such gene sequences may be encompassed withinthe claimed polynucleotides comprising (or consisting of) a nucleotidesequence according to type (c) above. Southern hybridization using thenucleotide sequences of type (a) (or fragments thereof) as probes, canidentify such related sequences in genomic, cDNA or mRNA libraries fromother bacteria. Alternatively, the use of PCR, with primers comprisingthe A. pleuropneumoniae sequences or fragments thereof, can be equallyas effective for identifying virulence genes across species boundaries.As a further alternative (which does not rely on hybridisation but stillidentifies homologous genes) complementation of an A. pleuropneumoniaemutant strain with a polynucleotide (e.g. DNA or mRNA) library fromanother species, can be used to identify genes having the same, or arelated biological activity. In short, polynucleotides of the invention,which comprise a nucleotide sequence according to type (c), may be foundin, or constitute, virulence genes of other bacteria related to A.pleuropneumoniae.

Identification of related virulence genes can lead to the production ofan attenuated strain of the other organism by mutation in that gene, andmay therefore be used in the production of vaccines and otherimmunological compositions. In yet further aspects, the presentinvention thus provides: (1) an attenuated bacterium (e.g. of thePasteurellaceae family) containing a mutation in a gene comprising anucleotide sequence which is capable of hybridising to any one of thenucleotide sequences defined by SEQ ID NO:1-56, under conditions ofmoderate to high stringency; (2) a composition (e.g. a vaccine)containing such a bacterium; and (3) the use of such a bacterium for themanufacture of a medicament (e.g. a vaccine) for the therapeutictreatment or prophylactic protection of an animal against infection bythe corresponding wild-type bacterium (or a different strain or serotypethereof).

As explained elsewhere herein, polynucleotides of the present inventionof types (a)-(d) may be e.g. DNA, RNA, or peptide nucleic acids, asdescribed for example in Corey, TIBTECH 15: 224-229 (1997). DNAsequences include those derived (extracted or amplified) from genomicDNA, or from cDNA, as well as wholly or partially chemically synthesizednucleotide sequences. Amplification may be effected by any method wellknown to those skilled in the art, e.g. by PCR.

“Chemically synthesized” as used herein refers to purely chemical, asopposed to enzymatic (e.g PCR-based) methods for producingpolynucleotides. “Wholly” synthesized polynucleotides are those producedentirely by chemical means, and “partially” synthesized polynucleotidesinclude those wherein only portions of the resulting sequence areproduced by chemical means.

The polynucleotides of the invention, or their gene products, may beribozymes. Such polynucleotides may comprise or consist of an (RNA)nucleotide sequence according to type (c) above, i.e. one which iscapable of hybridising to any of the nucleotide sequences recited in (a)or (b) under conditions of moderate to high stringency. For a review ofribozyme technology, reference is made to Gibson and Shillitoe, Mol.Biotech. 7: 125-137 (1997). A ribozyme can be utilized to inhibit thetranslation of mRNA (e.g. mRNA transcribed from virulence genes) in asequence specific manner, e.g. by: (i) the hybridization of acomplementary RNA sequence in the ribozyme to the target mRNA; and (ii)cleavage of the hybridized target mRNA through nuclease activityinherent to the ribozyme. The polynucleotides of the invention maytherefore be used to inhibit the expression of virulence genes of A.pleuropneumoniae or of other related e.g. Pasteurellaceae bacteria.Ribozymes can be identified by empirical methods, but they may bespecifically designed based on accessible sites on the target mRNA, e.g.on the mRNA transcription product of the nucleotide sequences defined bySEQ ID NO:1-56 (see also Bramiage et al, Trends in Biotech 16: 434-438(1998)).

Delivery of ribozymes to target cells, in order to control mRNAtranslation, may be accomplished using delivery techniques well known tothose skilled in the art. Exogenous delivery methods can include the useof targeting liposomes or direct local injection. Endogenous methodsinclude the use of viral vectors and of non-viral plasmids.

Certain polynucleotides of the present invention may also be used tomodulate the transcription of virulence genes in A. pleuropneumoniae,e.g. by means of oligonucleotide-directed triplet helix formation. For areview of this technique, reference is made to Lavrovsky et al.,Biochem. Mol. Med. 62: 11-22 (1997). Triplet helix formation isaccomplished using sequence specific oligonucleotides which hybridize todouble stranded DNA in the major groove (as defined in the Watson-Crickmodel). Hybridization of such sequence specific oligonucleotides canthereafter modulate the activity of DNA-binding proteins, including, forexample, transcription factors and polymerases. Preferred targetsequences for hybridization include transcriptional regulatory regionsthat modulate the expression of virulence genes. Oligonucleotides whichare capable of triplet helix formation are also useful for site-specificcovalent modification of target DNA sequences in virulence genes.Oligonucleotides useful for covalent modification are coupled to DNAdamaging agents as described for example in Lavrovsky, et al, Biochem.Mol. Med. 62: 11-22 (1997).

In yet another aspect, the present invention provides a vectorcomprising a polynucleotide according to the present invention. Thevector may be DNA or RNA in nature. It may contain one or more controlsequences operably linked to the polynucleotide, in order to promote itstranscription, and optionally enable its translation, in a suitable hostcell under appropriate conditions. Vectors may be plasmids, or viralvectors. They may be capable of autonomous replication. Controlsequences may be endogenous (i.e. found within the wild-type bacteriumfrom which the polynucleotide of the invention is derived, e.g. A.pleuropneumoniae) or they may be exogenous (e.g. a control sequencefound in a related bacterium, e.g in another member of thePasteurellaceae family). Control sequences include interalia promotersand transcription terminators.

Host cells containing (e.g. transformed, transfected, or electroporatedwith) a polynucleotide or vector of the present invention are furthercontemplated. Such host cells may be prokaryotic or eukaryotic innature, either stably or transiently transformed, transfected, orelectroporated with a polynucleotide or vector of the present invention,in a manner which permits the transcription of the polynucleotide, andoptionally enables translation of the resultant mRNA. Host cells of theinvention include bacterial, yeast, fungal, invertebrate, and mammaliancells. Viruses containing a polynucleotide or vector of the presentinvention are also contemplated.

Host cells of the present invention may be useful in methods for thelarge scale production of virulence polypeptides (where thepolynucleotide of the invention encodes a polypeptide sequence). Thecells are grown in a suitable culture medium under favourableconditions, and the desired polypeptides are isolated from the cells, orfrom the medium in which the cells are grown, by any purificationtechnique well known to those skilled in the art, e.g. by capture on anaffinity column. Such cells may be a valuable source of immunogen forthe development of antibodies which specifically bind to the virulencepolypeptide. Any suitable host cell may be used for the expression of apolynucleotide according to the present invention.

For this and other purposes, polynucleotides of the present inventionmay be manipulated using standard techniques well within the ambit ofthose skilled in the art, e.g. the techniques described in Sambrook etal, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y. (1989). By way of example,the polynucleotides of the invention may be amplified and cloned by e.g.PCR, e.g. using genomic DNA, cDNA or mRNA as a template. For ease ofinserting the cloned sequence into vectors, e.g. expression vectors, PCRprimers may be chosen so that the PCR-amplified sequence has arestriction enzyme site at the 5′ end which precedes the initiationcodon ATG, and a restriction enzyme site at the 3′ end after thetermination codon TAG, TGA, or TM. If desirable, the codons in thecloned sequence may be changed, without changing the amino acids,according to the codon preference of an intended host cell, e.g. E.Coli, as described by Grosjean and Fiers, Guide, 18: 199-209 (1982) andKonigsberg and Godson, Proc. Natl. Acad. Sci. (USA), 80: 687-691 (1983).Optimization of codon usage may lead to an increase in the expression ofthe gene product when produced in that host.

If a virulence polypeptide is to be produced extracellularly, e.g. inthe periplasm of a host bacterium, or in the culture medium surroundinga host cell, then the polynucleotide of the present invention may becloned without its initiation codon, and placed into an expressionvector behind an appropriate signal sequence. A number of signalsequences from both prokaryotes and eukaryotes are known to function inthe secretion of recombinant proteins. During the protein secretionprocess, the signal peptide may be removed by a signal peptidase toyield the mature protein.

To simplify the purification of virulence polypeptides, a purificationtag may be added to the 5′ and/or 3′ end of the polypeptide encodingsequence. Commonly used purification tags include a stretch of sixhistidine residues (U.S. Pat. Nos. 5,284,933 and 5,310,663), astreptavidin-affinity tag described by Schmidt and Skerra, ProteinEngineering, 6: 109-122 (1993), a FLAG peptide (Hopp et al.,Biotechnology, 6: 1205-1210 (1988)), glutathione S-transferase (Smithand Johnson, Gene, 67:31-40 (1988)), and thioredoxin (LaVallie et al.,Bio/Technology, 11: 187-193 (1993)). To remove these tags, a proteolyticcleavage recognition site may be inserted at the fusion junction.Commonly used proteases are factor Xa, thrombin, and enterokinase.

In a further aspect, the present invention provides a method ofproducing a virulence polypeptide encoded by a polynucleotide of theinvention. The method may comprise the steps of: (i) culturing a hostcell according to the present invention under conditions which permitthe expression of the polypeptide; and (ii) recovering and optionallyisolating the expressed polypeptide from the host cell, or from itssurrounding medium. Identification of the polynucleotides of the presentinvention readily makes available their encoded polypeptides, so suchpolypeptides can alternatively be chemically synthesised.

Polypeptides encoded by polynucleotides of the present invention (andvariants of such polypeptides) are a further aspect of the invention.Such polypeptides are “virulence polypeptides”, as discussed elsewhereherein. Compositions containing such polypeptides are furthercontemplated, and these include vaccines and other immunogeniccompositions. Such compositions may contain one or more adjuvants,carriers, diluents, vehicles or excipients, as discussed elsewhereherein.

Variants include biologically and/or immunologically active fragments ofthe polypeptide encoded by a polynucleotide of the invention, e.g.fragments of the polypeptide encoded by the polynucleotide sequencedefined in any one of SEQ ID NO.:1-56, fusions of the polypeptides orfragments thereof with e.g. heterologous polypeptides (thereby toproduce chimeras), and analogs of the polypeptides, e.g. polypeptides inwhich one or more amino acids has been deleted, replaced or added: (i)without loss of one or more of the biological activities and/orimmunological characteristics specific for the polypeptide; or (ii) withspecific disablement of a particular biological activity of thepolypeptide. Replacement may occur using conservative amino acids.

Virulence polypeptides having an additional methionine residue atposition −1 are contemplated, as are virulence polypeptides havingadditional methionine and lysine residues at positions −2 and −1.Variants of these types are particularly useful for recombinant proteinproduction in bacterial cell types. The invention also embraces variantshaving additional amino acid residues which result from the use ofspecific expression systems. For example, use of commercially availablevectors that express a desired polypeptide as a fusion protein withglutathione-S-transferase (GST) provide the desired polypeptide havingan additional glycine residue at position −1 after cleavage of the GSTcomponent from the desired polypeptide. Variants of polypeptides of thepresent invention also include those in which a leader or signalsequence has been removed, e.g. a mature form.

Variant polypeptides include those which have at least about 99%, atleast about 95%, at least about 90%, at least about 85%, at least about80%, at least about 75%, at least about 70%, at least about 65%, atleast about 60%, at least about 55%, and at least about 50% sequenceidentity and/or homology with a virulence polypeptide encoded by anucleotide sequence as defined in any one of SEQ ID NO.:1-56. Percentage“identity” of an amino acid sequence is defined herein as the percentageof amino acid residues in the candidate sequence that are identical withthe residues in the virulence polypeptide, after aligning both sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions aspart of the sequence identity. Percent “homology” of an amino acidsequence with respect to a virulence polypeptide encoded by any one ofSEQ ID NO:1-56 refers to the percentage of amino acid residues in thecandidate sequence that are identical with the residues in the virulencepolypeptide after aligning the two sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and alsoconsidering any conservative substitutions as part of the sequenceidentity.

By conservative substitution is meant exchange of one amino acid for anamino acid of the same type, the types being as follows: AliphaticNon-polar: G A P I L V Polar-uncharged sulphur: C M Polar-unchargedhydroxyl: S T Polar-charged acidic: D E N Q Polar-charged basic: K R HAromatic: F W Y

In a still further aspect, the present invention extends to antibodieswhich specifically recognize the polynucleotides or polypeptides of thepresent invention. Antibodies may be monoclonal or polyclonalantibodies. They include chimeric antibodies, e.g. humanized antibodies,e.g. complementary determining region (CDR)-grafted antibodies, as wellas fragments of antibodies (which may be chimeric) e.g. Fab, F(ab′)₂, Fdand single chain antibodies. They also include compounds having CDRsequences which specifically recognize a polynucleotide or polypeptideof the present invention. The invention also provides anti-idiotypeantibodies which specifically bind to antibodies of the presentinvention.

The term “specific for” indicates that the variable regions of theantibodies of the invention exclusively recognize a target polypeptideor polynucleotide (i.e. they are able to distinguish a singlepolypeptide or polynucleotide from related polypeptides orpolynucleotides). Binding affinities of at least about 10⁷ M⁻¹, at leastabout 10⁸ M⁻¹, or at least about 10⁹ M⁻¹ are contemplated. That is notto say that the antibodies will not also interact with other proteins(for example, S. aureus protein A or other antibodies in ELISAtechniques) through interactions with sequences outside the variableregion of the antibodies, and in particular, in the constant region ofthe molecule. Screening assays to determine binding specificity of anantibody of the invention are well known and routinely practised bythose skilled in the art. For a comprehensive discussion of such assays,reference may be made to Harlow et al. (Eds), Antibodies. A LaboratoryManual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988),Chapter 6.

According to a still further aspect of the present invention, materialsand methods are provided for identifying an anti-bacterial agent whichis capable of modulating the function of an A. pleuropneumoniaevirulence gene identified by the present invention, or of a homologousgene in a related species. Agents which are capable of modulating thefunction of such virulence genes are likewise contemplated per se. Moreparticularly, methods of the present invention include screeningpotential agents for their ability to interfere with the expressionand/or biological activity in a host bacterium of the gene productsencoded by the nucleotide sequences set forth in any one of SEQ IDNO:.1-56. Agents that interfere with the expression of virulence geneproducts include (but are not limited to) anti-sense polynucleotides andribozymes containing nucleotide sequences which are homologous to anyone of the nucleotide sequences of SEQ ID NO:.1-56 (such that they bindto its complement). The invention further embraces methods of modulatingthe transcription of such virulence genes through the use ofoligonucleotide-directed triplet helix formation.

Compositions containing an anti-bacterial agent identified in accordancewith the present invention are a still further aspect of the presentinvention, in addition to a method of treating an animal suffering froma Pasteurellaceae (e.g. an A. pleuropneumoniae) infection byadministration of such an anti-bacterial agent or composition. Theinvention also embraces the use of such an agent or composition for themanufacture of a medicament for the treatment of a Pasteurellaceaeinfection, e.g. an A. pleuropneumoniae infection and/or porcinepleuropneumonia.

Agents that interfere with the function of virulence genes encompass:(i) non-functional variants of the virulence gene products which may becapable of out-competing the wild-type product for its physiologicalbinding sites; and (ii) binding partners of the virulence gene products,which sequester and optionally destroy said products. Agents alsoinclude allosteric enzyme inhibitors, in the case where the virulencegene product is an enzyme.

Inhibitory agents may be identified by means of high throughputscreening assays using the polynucleotides of the present invention ortheir encoded products (which may be RNA polynucleotides orpolypeptides). Where the screen employs polypeptide gene products havingenzymatic activity, assays are established which are based on thatactivity, and potential agents screened for an ability to modulate (e.g.inhibit) the activity. For virulence gene products which exert theirfunction by interacting with a polypeptide or nucleic acid, bindingassays are established to measure such interaction, and potential agentsare screened for an ability to modulate, e.g. to inhibit theinteraction.

In one example of a binding assay, the virulence gene product isimmobilized on a solid support, and an interaction with a(physiological) binding partner is assessed in the presence and absenceof a putative inhibitor compound. In another example, interactionbetween the virulence gene product and its binding partner is assessedin a solution assay, both in the presence and absence of a putativeinhibitor compound. In both assays, an inhibitor is identified as acompound that decreases binding between the virulence gene product andits binding partner. Assays for use when the virulence gene product andits binding partner are both polypeptides include variations of thedi-hybrid assay, wherein an inhibitor of protein/protein interaction isidentified by detection of a positive signal in a transformed ortransfected host cell, as described for example in publishedInternational patent application WO 95/20652.

Candidate inhibitors contemplated for use in the methods of the presentinvention include compounds selected from libraries of potentialinhibitors. A number of different libraries can be used as a source ofsmall molecule modulators, including: (1) chemical libraries; (2)natural product libraries; and (3) combinatorial libraries comprised ofrandom peptides, oligonucleotides or organic molecules.

Chemical libraries consist of structural analogs of known compounds.Natural product libraries are collections of microorganisms, animals,plants, or marine organisms which are used to create mixtures forscreening by: (i) fermentation and extraction of broths from soil, plantor marine microorganisms; or (ii) extraction of plants or marineorganisms. Natural product libraries are further discussed in e.g.Science 282: 63-68 (1998). As for combinatorial libraries, these arecomposed of large numbers of peptides, oligonucleotides, or organiccompounds as a mixture. They are relatively easy to prepare bytraditional automated synthesis methods, PCR, cloning, or proprietarysynthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. For a review of combinatorialchemistry libraries, reference may be made to Myers, Curr. Opin.Biotechnol. 8:701-707 (1997).

Still other candidate inhibitors, which are contemplated for use in themethods of the invention, may be specifically designed. These includesoluble forms of the binding partners of virulence polypeptides. Theterm “binding partners” as used herein encompasses antibodies (althoughin certain embodiments these are excluded) including fragments andderivatives thereof, and modified compounds comprising antibody domainsthat specifically bind to the target gene product.

When a polypeptide binding partner for a virulence gene product is notknown, assays that identify physiological, or non-physiological andhence sequestering binding partners may be employed, e.g. assays usingaffinity columns on which the virulence gene product is immobilised.Alternatively, binding interactions between polypeptides may beidentified using the yeast two-hybrid system as described in Fields andSong, Nature, 340: 245-246 (1989), and Fields and Sternglanz, Trends inGenetics, 10: 286-292 (1994). Other assays which may be used to searchfor agents that bind to a polypeptide virulence gene product includethat described in U.S. Pat. No. 5,585,277, which relies on the principlethat proteins generally exist as a mixture of folded and unfoldedstates, and continually alternate between the two states. When a testligand binds to the folded form of a target virulence polypeptide, thetarget protein molecule bound by the ligand remains in its folded state.Thus, the folded target protein is present to a greater extent in thepresence of a test ligand which binds the target protein, than in theabsence of a ligand. Binding of the ligand to the target protein can bedetermined by any method which distinguishes between the folded andunfolded states of the target protein. The function of the targetprotein need not be known in order for this assay to be performed.Virtually any agent can be assessed by this method as a test ligand,including, but not limited to metals, polypeptides, including proteins,lipids, polysaccharides, polynucleotides and small organic molecules.

Another method for identifying ligands of a virulence gene polypeptideis that described in Wieboldt et al, Anal. Chem., 69: 1683-1691 (1997).That technique screens combinatorial libraries of 20-30 agents at a timein solution phase for binding to the target virulence polypeptide.Agents that bind to the target are separated from other smaller librarycomponents by centrifugal ultra-filtration. The specifically selectedmolecules that are retained on the filter are subsequently liberatedfrom the target protein and analysed, e.g. by HPLC and pneumaticallyassisted electrospray (ion spray) ionization MS. This procedure selectslibrary components with the greatest affinity for the target protein,and is particularly useful for libraries of small molecules.

Binding agents identified by the initial screens maybe evaluated fortheir effect on virulence in vivo. Agents that interfere with bacterialvirulence can prevent the establishment of an infection, or reverse theoutcome of an infection once it is established. Binding agents may beincluded in compositions containing pharmaceutically acceptableadjuvants, excipients, diluents, carriers or vehicles, as describedelsewhere herein.

Numerous additional aspects and advantages of the invention will nowbecome apparent to those skilled in the art upon consideration of thefollowing detailed description of the invention which describespresently prepared embodiments thereof.

EXPERIMENT 1

A. Materials and Methods

1. Bacterial Strains and Growth Conditions

A spontaneously nalidixic acid-resistant derivative of the virulentActinobacillus pleuropneumoniae serotype 1 strain 4074 was selectedusing conventional methods and designated 4074 Nal®. The virulence ofthis strain was verified by passage in pigs. A. pleuropneumoniae strainswere propagated at 37° C. with 5% CO₂ on brain-heart infusion (BHI)plates supplemented with 10% Levinthal's base (BHI_(L)), or at 37° C. inColombia broth with 5 μg/ml β-nicotinamide adenine dinucleotide (NAD)and 11 mM CaCl₂.

The E. coli strains used in this study were: S17 λ pir [recA thi prohsdR^(−M) ⁺ RP4::2-Tc::Mu::Km Tn 7 lysogenized with λ pir phage] asdescribed in Miller and Mekalanos (1988) J. Bacteriol. 170, pp.2575-2583; CC118 λ pir [Δ(are-leu) araD Δ lacX74 galE galK phoA20 thi-1rpsE rpoB argE recA1 lysogenized with λ pir phage] as described in deLorenzo et al, (1990) J. Bacteriol. 172, pp. 6657-6667; and XL1-Blue(recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacI^(q)ZΔM15 Tn10 (Tet′)]). E. coli strains were maintained in Luria-Bertani(LB) medium with appropriate antibiotics used at the followingconcentrations: ampicillin 100 μg/ml; kanamycin 100 μg/ml; nalidixicacid 20 μg/ml; tetracycline 100 μg/ml.

2. DNA Manipulations, Amplification, Labelling, and Hybridisation ofTags

Chromosomal DNA was isolated from A. pleuropneumoniae using a QIAampmini DNA kit (Qiagen) according to the manufacturer's instructions.Plasmid DNA was isolated from E. coli using a Qiagen plasmid midi kit,again according to the manufacturers instructions. DNA manipulation wasperformed according to standard techniques as described in Sambrook etal, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y. (1989). Probes for southernblots were labelled with [digoxigenin (DIG)-11]-dUTP using a PCR DIGProbe Synthesis Kit (Roche) as described by the manufacturer.Amplification and labelling of DNA signature-tags was performed asdescribed in Hensel et al (1995) Science 269 pp. 400-403. Tags wereinitially amplified from genomic DNA by means of PCR using the followingprimers: P6 (5′-CGACTACAACCTCAAGCT-3′) P7 (5′-CGACCATTCTAACCAAGC-3′)

The amplified tags were purified on a 2% Nusieve GTG agarose gel andradiolabelled with [³²P]-dCTP in a second PCR, again using P6 and P7.The cycling conditions for both PCRs were as follows: denaturation andenzyme activation 12 min at 95° C., followed by 20 cycles of: 95° C. for30 s, 50° C. for 45 s and 72° C. for 10 s. Reactions were performedusing 1 unit of AmpliTaq Gold (Perkin-Elmer) polymerase in aPerkin-Elmer 2400 thermocycler. Prior to use, the invariant flanking DNAwas separated from the variable signature-tag by digestion with HindIIIfor 18 h at 37° C.

For colony blots, E. coli S17 λ pir strains harbouring each of thepLOF/TAG 1-48 plasmids were grown to mid-exponential phase and 100 μl ofeach culture was transferred to the wells of a microtitre dish. Bacteriawere transferred from the microtitre dish to a nylon membrane (Hybond N)using a replica plater (Sigma) and the inoculated membrane placed on thesurface of a dried LB agar plate containing ampicillin (100 μg/ml).After overnight growth at 37° C., the membranes were treated with 1.5 MNaCl, 0.5 M NaOH for 10 min. DNA was fixed to the membrane bymicrowaving at maximum power for 30 s and bacterial debris removed bywashing in 2×SSC. Pre-hybridisation and hybridisation were performed at65° C. in Rapid-hyb buffer (Amersham) for 1 h and overnight,respectively. Blots were washed according to standard procedures. Toidentify potentially attenuated mutants, hybridisation signals on a blotprobed with labelled tags prepared from the input pool (inoculum) werecompared to identical blots hybridised with labelled tags prepared fromthe corresponding output pools from at least two different animals.

3. Construction of DNA Signature-Tags and pLOF/TAG 1-48

Double-stranded DNA signature tags were obtained by PCR amplification ofthe variable oligonucleotide pool RT1 (Hensel et al., 1995 Science 269pp. 400-403) using the following primers: P3SAL(5′-CGCCATGTCGACCATTCTAACCAAGCTT-3′) P5SAL(5′-CGCCTAGTCGACTACAACCTCAAGCTT-3′)

each of which contains recognition sites for the restrictionendonuclease SalI at its 5′ terminus. To facilitate the cloning of thesignature-tags in plasmid pLOF/Km, the plasmid was modified toincorporate a unique SalI site. This was done by annealing the followingcomplementary oligonucleotides: SALTOP (5′-GTCGACCCT-3′) and SALBTM(5′-GTCGACAGG-3′)to give a double-stranded linker containing a SalI site flanked bySfiI-compatible ends. Plasmid pLOF/Km was digested with SfiI, treatedwith calf intestinal alkaline phosphatase (CIAP), and ligated to thephosphorylated double-stranded linker to give plasmid pLOF/Sal. ThePCR-amplified double-stranded DNA signature-tags were digested withSalI, gel purified, and cloned into the SalI-digested, CIAP-treated,pLOF/Sal to generate a library of uniquely tagged pLOF/TAG plasmids.

Individual transformants in E. coli strain CC118 λ pir were screened bycolony blot hybridisation with their corresponding [³²P]-dCTP-labelledtags to identify 100 tags that amplified and labelled efficiently. Thesetags were then tested for an absence of cross-hybridisation and 48plasmids (pLOF/TAG1-48) were selected for further use.

4. Construction of the Signature-Tagged Mutant Bank

The 48 signature-tagged mini-Tn10 transposons carried on plasmidspLOF/TAG 1-48 were separately introduced into A. pleuropneumoniae byconjugation. A. pleuropneumoniae strain 4074 Nal® and E. coli S17 λ pirharbouring each of the pLOF/TAG 1-48 plasmids were grown overnight at37° C. to the stationary phase of growth. Bacteria were harvested bycentrifugation for 15 minutes at 3,000 rpm and washed twice with 10 mMMgSO₄. Approximately equal numbers of A. pleuropneumoniae recipient andE. coli donor cells were mixed, concentrated, and spread onto 0.22 μMfilters. Filters were placed onto BHI_(L) plates containing 1 mM IPTGand incubated at 37° C. with 5% CO₂ for 5 hours. Thereafter, thebacteria were removed into 5 ml sterile PBS and aliquots plated ontoBHI_(L) plates supplemented with nalidixic acid (20 μg/ml) and kanamycin(100 μg/ml). Four separate matings were performed for each of the 48signature-tagged mini-Tn10 transposons. Transconjugant mutants werescreened for susceptibility to ampicillin, thereby to eliminate strainscarrying co-integrants of the suicide vector, inserted into thechromosome. In total 2064 transposon mutants from 48 individual matingswere isolated and arranged into 43 pools of 48 mutants. Mutants werestored in 1.5 ml cryotubes with 15% glycerol at −80° C.

5. Infection Studies

Individual strains in each pool of 48 A. pleuropneumoniaesignature-tagged mutants were grown separately until the mid-exponentialphase of growth. At an OD₆₀₀ of approximately 0.3, the individual mutantstrains were combined and the pooled culture was diluted in HEPES toapproximately 2×10⁶ cfu/ml. Large White Cross piglets, of 6-10 weeks ofage and determined to be A. pleuropneumoniae free by bacteriological andserological analyses, were inoculated intra-tracheally with 3 ml of thediluted culture. The number of cfu in the inoculum was verified byviable counts after plating serial dilutions of the inoculum onselective BHI_(L). Each mutant pool was used to infect at least twoanimals.

At approximately 24 h post-infection, surviving animals were euthanisedand the lungs were removed. Surviving bacteria were recovered frominfected lungs by either: (i) extensive lavage with Hanks bufferedsaline solution (HBSS); or (ii) homogenising the lungs in 500 ml HBSS.Aliquots of the bacterial suspension were plated on selective media, andfollowing growth overnight at 7° C. with 5% CO₂, approximately 10,000colonies were pooled (the “output pool”) and total chromosomal DNA wasprepared.

6. Competitive Indices

To determine the in vivo competitive index (CI) of individual STMmutants, mutant and wild-type bacteria were grown in Colombia broth with5 μg/ml NAD and 11 mM CaCl₂ to an OD₆₀₀ of 0.3. The cultures were thencombined to give equal numbers of mutant and wild-type organisms,diluted to 2×10⁶ cfu/ml, and inoculated intra-tracheally into at leasttwo animals. The ratio of mutant to wild-type bacteria in the inoculumwas verified by viable counts after plating serial dilutions of theinoculum in parallel on BHI_(L)+20 μg/ml nalidixic acid (mutant andwild-type), and BHI_(L)+20 μg/ml nalidixic acid+100 μg/ml kanamycin(mutant only). Approximately 24 h post-infection, surviving animals wereeuthanised and the lungs were removed. The lungs were homogenised in 500ml HBSS and aliquots were plated on selective media (see above) torecover surviving bacteria. The in vivo CI was calculated as the outputratio of mutant to wild-type organisms divided by the input ratio ofmutant to wild-type organisms. To determine in vitro CIs, mutant andwild-type organisms were grown to mid-exponential phase, mixed in equalnumbers, and the combined culture incubated, with agitation, at 37° C.for a further 3 h. Samples were taken at regular intervals, diluted, andplated on selective BHI_(L) as before, and the in vitro CI wascalculated as described above.

7. Virulence Gene Identification and DNA Sequencing

To identify the in vivo attenuating lesions, DNA flanking the site oftransposon insertion was cloned or amplified by inverse PCR. Forcloning, chromosomal DNA was digested with a panel of restrictionenzymes, separated by agarose gel electrophoresis, transferred to anylon membrane, and probed in a Southern blot with a DIG-labelled probespecific for the Tn10 kanamycin gene. Suitably sized fragmentshybridising to the probe were excised from a gel, purified and clonedinto appropriately digested pBR322. Transformants in E. coli strainXL1-Blue were selected on LB agar containing 100 μg/ml kanamycin.

To facilitate sequencing with the Tn10 specific oligonucleotide primer:BS401 (5′-GACAAGATGTGTATCCACC-3′)the cloned insert was further sub-cloned into plasmid pBCKS(Stratagene). Inverse PCR amplification of DNA flanking the site oftransposon insertion was performed as described in Fuller, (2000)Microb. Pathog. 29, pp. 25-38: chromosomal DNA was digested with SspI(which cuts once in the transposon), diluted to 150 ng/ml, andself-ligated for 15 min using the Rapid DNA Ligation Kit (Roche). Theligated DNA was then amplified using the primer pairs BS401+TEF48, andBS401+TEF62 (Fuller, 2000 Microb. Pathog. 29, pp. 25-38). Reactionconditions for inverse PCR were as previously described (Fuller, 2000Microb. Pathog. 29, pp. 25-38). The resultant amplicons were purifiedfollowing agarose gel electrophoresis using a QIAQUICK gel extractionkit (Qiagen). DNA sequencing reactions were performed using the BigDyeterminator cycle sequencing kit (Perkin-Elmer). Sequences from each ofthe attenuated mutants were used to query the non-redundant public DNAand protein databases using the BLAST set of similarity search programs(Altschul et al 1990 J. Mol. Biol. 15, pp. 403-410).B. Results and Discussion1. Generation of Signature-Tagged Mutants of A. pleuropneumoniae.

The plasmid pLOF/Km, which is functional in A. pleuropneumoniae, wasmodified to accept signature-tags by the insertion of a unique SalI sitein the mini-Tn10 element. PCR amplified random signature-tags werecloned into the SalI site, and a bank of approximately 30,000 taggedplasmids in E. coli CC118 λ pir was obtained. A subset of thesetransformants was screened by colony blot with their correspondingradiolabelled tags, and signature-tagged plasmids with stronghybridisation signals were selected. Plasmids with cross-hybridisingtags were eliminated, and a pool of 48 unique signature-tagged plasmidswas assembled. These plasmids (pLOF/TAG 148) were transformed into E.coli S17 λ pir, and independently transferred to A. pleuropneumoniaestrain 4074 Nal® by conjugation.

Following the elimination of ampicillin-resistant plasmid cointegrants(0.5-1%), 2064 transposon mutants from 48 individual matings wereisolated, and then arranged into 43 pools of 48 mutants. Southern blotanalysis of ten randomly chosen exconjugants with a mini-Tn10-specificprobe confirmed single, random insertion of the tagged transposon (datanot shown). However, subsequent sequence analysis of attenuated clonesrevealed that mini-Tn10 insertion in A. pleuropneumoniae was notentirely random.

2. Signature-Tagged Screen of A. pleuropneumoniae mutants in Pigs

Signature-tagged A. pleuropneumoniae mutants were screened using aporcine intra-tracheal infection model to identify genes involved invirulence. Bacteria were instilled directly into the trachea as previousstudies have shown that aerosol particles produced by sneezing are smallenough to penetrate into the lower respiratory tract, obviating the needfor colonisation of the upper respiratory tract (Kaltreider H B. inImmunologic and infectious diseases in the lung. (1976) pp. 73-97,Kirkpatrick & Reynolds (Eds) Marcel Dekker, New York).

A range of experimental factors including the infecting dose, and themethod of recovery were investigated before we could reproduciblyrecover the majority of individual mutants following in vivo selectionin the porcine host. Using challenge doses of 10³, 10⁴, or 10⁵ cfu, forexample, there was a poor correlation between the infecting dose and theappearance of clinical or morphological signs of disease. Moreover, whendifferent pigs were infected with the same pool, the hybridisationpatterns of the post-passage pool suggested that different clones weremissing after passage of a given pool through different animals (datanot shown). This inconsistency was overcome by increasing the challengedose to 10⁶-10⁷ cfu/pig. In an earlier STM study of A. pleuropneumoniaeinfection in pigs [Fuller, 2000 Microb. Pathog. 29, pp. 39-51], bacteriawere recovered by lung lavage following experimental infection. However,this method was found to be unreliable, yielding essentially randomrecovery of individual signature tags. In contrast, we observedconsistent recovery of viable bacteria following homogenisation of theentire lung in HBSS. Optimisation of these two crucial parametersresulted in the recovery of the majority of individual clones (>90%,20-48 h post infection), with excellent reproducibility between animals(see FIG. 1; compare A and B).

In total, 2064 transposon mutants (43 pools of 48 mutants) were screenedfor attenuation in vivo, each pool being inoculated into at least twodifferent animals. Surviving bacteria were recovered by homogenisationof lungs in sterile HBSS. This initial screen identified 550 mutantspresent in the input pool but missing or significantly reduced in theoutput pools. These potentially attenuated mutants were reassembled intonew pools and each new pool was used to infect a further two animals.Following this second screen, 105/410 mutants were found to beconsistently absent from the output pools, and these were retained forfurther analysis.

3. Identification and Analysis of A. pleuropneumoniae Virulence Genes.

The nucleotide sequence of the DNA flanking the site of transposoninsertion was obtained for each of the 105 attenuated mutants asdescribed, and used to search the GenBank databases for homologousgenes. The results of this analysis are shown in Table 1, with the genesassigned to one of five functional categories: cell surface, transport,metabolism, stress responses, and regulation. Insertions in genesencoding proteins with no significant homology to proteins in thedatabases, or homologous to proteins or predicted proteins of unknownfunction, are categorised as “unknown”. The 105 attenuated mutantscontained transposon insertions in 53 individual genes and a number ofhotspots for Tn10 insertion were identified. In particular, sevenidentical insertions were independently isolated in the putative rfbPgene (Tn insertion at codon 444), 3 of 4 insertions in the A.pleuropneumoniae fur gene were located at codon 128. Interestingly, theDNA sequences flanking the 3 hotspots which were identified in thisstudy are neither similar to each other, nor to the consensus sequencefor Tn10 insertion into A. pleuropneumoniae that has been previouslybeen identified (Fuller et al (2000) Microb. Pathog. 29, pp. 25-38).

(i) Cell Surface

Mutations in capsule and lipopolysaccharide (LPS) genes are known toattenuate A. pleuropneumoniae (Rioux et al., Can. J Microbiol. 45: pgs1017-1026, 1999) (Rioux et al., Microb Pathog 28: pgs 279-289, 2000;Ward et al., Infect Immun 66: pgs 3326-3336, 1998). Mutant 9C2 containsa transposon insertion in the cpxC gene that encodes a protein involvedin capsule export in A. pleuropneumoniae (Ward and Inzana, 1997). Thismutant was used as a control (known attenuated) strain the in vivo CIexperiments. Five genes involved in the synthesis of O-antigen of A.pleuropneumoniae serotype 1 were identified in this STM screen. Mutantswith inserts in genes 9 (mutant 10B11), 12 (mutant 25B7), 15 (mutant15A9), 17 (mutant 12D5), and 18 (mutant 21B8) of the O-antigen operon(Labrie et al., 2002, J. Endotox Res. 8: 27-38) were all attenuated.Another mutant, 9B7, has the transposon insertion 20 bases upstream ofthe A. pleuropneumoniae galU gene that encodes a protein involved insynthesis of the LPS core. Mutation of galU was previously shown toattenuate A. pleuropneumoniae (Rioux et al., 1999). Finally, mutant 26A9contains an insertion in a gene homologous to IcbB, a putativebifunctional polymerase implicated in capsule or LPS biosynthesis in N.meningiditis serogroup L.

Strain 4D4 contains a transposon insertion in a gene encoding a putativelipoprotein. Strain 4D4 shows greatest homology to an unknown proteinfrom P. multocida, but it is similar to lipoproteins and putativelipoproteins of H. influenzae and H. somnus [Theisen (1993) Infect Immun61: 1793-1798]. H. somnus LppB binds Congo red dye (a structuralanalogue of heme) and has been suggested to be a virulence factor, butthis has not been formally proven [Theisen (1993) Infect Immun61:1793-1798]. Finally, strain 17A4 contains an insertion in an ORF withhomology to the P2 porin protein of H. influenzae [Duim et al (1993)Microb. Pathog. 14, pp. 451-462]. The P2 protein constitutesapproximately one half of the total outer membrane protein of H.influenzae and represents an important target of the immune response tonon-typeable H. influenzae infection. [Yi et al (1997) Infect. Immun.65,150-155].

(ii) Metabolism

Strain 10B12 contains a transposon insertion in an ADP-ribosepyrophosphatase (adpP) gene homologue. Regulation of cellular levels ofADP-ribose (ADP-ribose is derived from NAD) is important in preventingnon-enzymatic ADP-ribosylation of proteins. The E. coli, ADP-ribosepyrophosphatase catalyses the hydrolysis of ADP-ribose to ribose-5-P andAMP, compounds that can be recycled as part of nucleotide metabolism[Gabelli et al (2001) Nat Struct. Biol. 8, pp. 467-472].

Strain 10A11 carries an insertion in an argininosuccinate synthase,argG, gene homologue. Argininosuccinate synthase catalyses theconversion of citrulline and aspartate into argininosuccinate, linkingthe urea cycle and the citric acid cycle via the so-calledaspartate-argininosuccinate shunt. The aspartate-argininosuccinate shuntprovides a metabolic link between the pathways by which amino groups andthe carbon skeletons of amino acids are processed. (Glansdorff (1996)Biosynthesis of arginine and polyamines. In Escherichia coli andSalmonella (Vol. 1) (Neidhardt, F. C. et al., eds), pp. 408-433, ASMPress).

In strain 33C7, the attenuating lesion was found to occur in a genehomologous to H. influenzae atpA, encoding the F₁ alpha subunit of F₁F₀ATP synthase. ATP synthase is responsible for utilising the protonelectrochemical gradient across the cytoplasmic membrane for ATPsynthesis in cells growing aerobically or anaerobically. All eightsubunits of the bacterial enzyme are encoded by the atp operon. In P.multocida the atpA gene is flanked by atpH and atpG, and while the geneorder in A. pleuropneumoniae is unknown, both atpH and atpG have beenimplicated previously in the pathogenesis of A. pleuropneumoniaeinfection [Fuller (2000) Microb. Pathog. 29, pp. 39-51].

In strain 0A7, the transposon has inactivated the pntB gene, encodingthe beta subunit of NAD(P) transhydrogenase. Transhydrogenase is anintegral membrane protein composed of two subunits, α and β, organisedas an α₂β₂ tetramer. Transhydrogenase couples the redox reaction betweenNAD(H) and NADP(H) to the translocation of protons across the innermembrane (Penfound & Foster (1996) Biosynthesis and recycling of NAD. InEscherichia coli and Salmonella (Vol. 1) (Neidhardt, F. C. et al., eds),pp. 408-433, ASM Press). It plays a key role in energy metabolism,biosynthesis and detoxification.

In strain 19D5, the transposon has inserted in the A. pleuropneumoniaemrp gene. The partial A. pleuropneumoniae mrp gene sequences availableare highly homologous to mrp genes from H. influenzae and E. coli. In H.influenzee, the mrp gene has been implicated in the biosynthesis of theGala(1-4)βGal component of LPS, while in E. coli mutations in mrp affectthe synthesis of thiamine [High et al (1996) FEMS Microbiol. Lett. 145,pp. 325-331; Petersen (1996) J. Bacteriol. 178, pp. 5676-5682]. In A.pleuropneumoniae, the mrp gene is located approximately 1 kb upstreamfrom the apxIVA gene, encoding the in vivo-induced RTX toxin, ApxIV[Schaller (1999) Microbiology 145: 2105-2116].

Strain 29B11 contains a transposon insertion in a napB gene homologue,encoding the smaller, dihaem cytochrome c, subunit of heterodimericperiplasmic nitrate reductase (NAP). In H. influenzae (and Pseudomonassp) NAP is the sole nitrate reductase and may function to supportanaerobic growth in the presence of nitrate [Brige (2001) Biochem. J.356, pp. 851-858; Fleischmann (1995) Science 269, pp. 496-512; Bedzyk(1999) J Bacteriol 181: 2802-2806]. In E. coli the situation is morecomplex with two membrane bound respiratory nitrate reductases inaddition to NAP [Moreno-Vivian (1999) J Bacteriol 181: 6573-6584]. Thereis evidence to suggest that NAP is a dissimilatory enzyme used for redoxbalancing. In addition, NAP has been suggested to play a role inadaptation to anaerobic metabolism after transition from aerobicconditions, the utilisation of alternate reductants, or even as aself-defense mechanism forming high nitrite levels to inhibit the growthof competing bacteria [Moreno-Vivian (1999) J Bacteriol 181: 6573-6584].Consistent with a role in anaerobic adaptation, transcription of theSalmonella typhimurium napB gene is induced, in an Fnr-dependent manner,in cells exposed to anaerobic conditions [Wei and Miller (1999) J.Bacteriol. 181, pp. 6092-6097].

Strain 17B8, contains an insertion in a gene homologous to H. influenzaeccmH, required for the assembly of C-type cytochromes, including NAP, inenteric bacteria [Thony-Meyer (1995) J. Bacteriol. 177, pp. 4321-4326;Kranz (1998) Mol Microbiol 29, pp. 383-396]. CcmH is encoded by thefinal gene of the ccm operon and with CcmF and CcmG is believed tocatalyse the reduction of disulphide bonds in the cytochrome capoprotein and to facilitate haem attachment [Kranz (1998) Mol Microbiol29, pp. 383-396]. While in E. coli the nap and ccm operons arecontiguous and may be cotranscribed [Grove (1996) Mol Microbiol 19, pp.467-481] they are located at separate loci in both H. influenzae and P.multocida [Fleischmann (1995) Science 269, pp. 496-512; May (2001) ProcNatl Acad Sci USA 98, pp. 3460-3465].

Strain 4B9 contains a transposon insertion in a moaA gene homologue. InE. coli the moaA-E genes are required for the synthesis of molybdopterinfrom GTP [Rivers (1993) Mol Microbiol 8: 1071-1081]. Molybdopterin(molybdopterin guanine dinucleotide) is the principal cofactor found inprokaryotic molybdoproteins including periplasmic nitrate reductase.Expression of the moa genes is induced by anaerobiosis [Anderson (2000)J. Bacteriol. 182, pp. 7035-7043] and Salmonella typhi moaA mutants areimpaired in their ability to replicate within epithelial cells[Contreras (1997) Microbiology 143, pp. 2665-2672].

Mutant 29B12 contains an insertion in the A. pleuropneumoniae recR genehomologue. The RecR protein is involved in the RecF pathway ofrecombinatorial DNA repair: recR mutants are deficient at filling indaughter strand gaps in newly synthesised DNA [Kuzminov (1999) MicrobiolMol Biol Rev 63, pp. 751-813]. Mutations in recR delay induction of theSOS response in E. coli and such mutants are sensitive to UV-irradiationand mitomycin C [Keller (2001) Mutat Res 486, pp. 21-29].

In strain 26A10, the transposon has inserted in a gene with extensivehomology to thrC of H. influenzae. The thrC gene encodes threoninesynthase, the final enzyme in the biosynthetic pathway leading to theproduction of threonine (Patte (1996) Biosynthesis of threonine andlysine. In Escherichia coli and Salmonella (Vol. 1) (Neidhardt, F. C. etal., eds), pp. 408-433, ASM Press). Mutations in a Streptococcuspneumoniae thrC homologue attenuate the virulence of this organism in amurine model of pneumoniae [Lau (2001) Mol Microbiol 40, pp. 555-571].

In strain 26D12, the transposon has inactivated a GMP synthase, guaA,gene homologue. GMP synthase catalyses the synthesis of GMP from XMP aspart of the purine biosynthetic pathway. Mutations in the guaBA operonattenuate the virulence of a variety of enteric pathogens including E.coli, S. flexneri, and S. typhi, and a ΔguaBA S. typhi derivative showspromise as a live attenuated vector [Wang (2001) Infect Immun 69, pp.4734-4741; Noriega (1996) Infect Immun 64, 3055-3061; Russo (1996) MolMicrobiol 22, pp. 217-229]. Moreover, previous STM screens inStreptococcus agalactiae and P. multocida have implicated guaA and guaB,respectively, in growth in vivo and in pathogenesis [Jones (2000) MolMicrobiol 37, pp. 1444-1455; Fuller (2000) Microb Pathog 29, pp. 39-51].

Mutant 27A12 contains a transposon insertion in a gene previouslyidentified in A. pleuropneumoniae as tonB (Tonpitak et al., Infect Immun68: 1164-1170 2000) (now referred to as tonB1). This gene is foundimmediately upstream of, and is be co-transcribed with, exbB, exbD, andthe transferrin binding protein (tbp) genes. In contrast, strain 0F6contains a transposon insertion in an ORF encoding a protein withhomology to TonB proteins from P. multocida and Haemophilus sp. (nowdesignated tonB2). Moreover, sequence analysis of the region upstreamfrom the transposon insertion in 0F6 revealed the presence of distinctexbB and exbD gene homologues, suggesting that A. pleuropneumoniae, incommon with other bacterial pathogens including V. cholerae and P.aeruginosa, possesses two independent TonB systems (Occhino et al., MolMicrobiol 29: 1493-1507 1998; Zhao and Poole, FEMS Microbiol Lett184:127-132 2000).

Strain 35D11 contains an insertion in a gene homologous to H. influenzaedsbA. DsbA is a small periplasmic protein and a member of thethioredoxin superfamily [Debarbieux (1999) Cell 99, pp. 117-119].Mutations in dsbA are pleiotropic, affecting the production of a varietyof translocated proteins including bacterial virulence factors,components of type III secretion machinery, and c-type cytochromesincluding NAP [Yu and Kroll (1999) Microbes Infect 1, pp. 1221-1228].Mutations in dsbA and dsbB have been implicated in the virulence ofShigelia flexneri [Yu (1998) Infect Immun 66, 3909-3917] and P.multocida [Fuller (2000) Microb Pathog 29, pp. 25-38], respectively.

In strain 26C3, the transposon has inactivated a glutamyl-tRNAreductase, hemA, gene homologue while in strain 26D5 the transposon hasinserted in a putative uroporphrinogen decarboxylase uroD/hemE genehomologue. Glutamyl-tRNA reductase catalyses the reduction of glutamateto glutamate 1-semialdehyde, the first committed step unique to theformation of δ-aminolevulinate (ALA) from glutamate. Downstream from ALAformation, uroporphrinogen decarboxylase catalyses the decarboxylationof uroporphrinogen III to yield coproporphrinogen, the first step in theconversion of uroporphrinogen III to protoheme via protoporphyrin IX.Hemes, including protoheme, are key components of the electron transferapparatus and in addition play important roles as enzyme prostheticgroups in mineral nutrition and oxidative catalysis (Beale, Biosynthesisof hemes. In Escherichia coli and Salmonella (Vol. 1) (Neidhardt, F. C.et al., eds), pp. 408-433, ASM Press).

In strain 15A11, the transposon has inserted into a gene homologous toP. multocida visC. The protein encoded by visC encodes a putativemonooxygenase belonging to the UbiH/CoQ6 family. UbiH is required forthe biosynthesis of ubiquinone [Nakahigashi (1992) J. Bacteriol. 174,pp. 7352-7359], a prenylated benzoquinone that functions in therespiratory electron chain in the plasma membrane of prokaryotes.Ubiquinone and protoheme also function in the formation of disulfidebonds in periplasmic proteins by facilitating the oxidation of DsbA byDsbB [Kobayashi (1997) Proc Natl Acad Sci USA 94, pp. 11857-11862].

Strain 29A10 contains a transposon insertion in a gene homologous toprfC of H. influenzae. The prfC gene encodes peptide chain releasefactor 3 (RF3), a GTPase that in the presence of GTP catalyses theremoval of RF1 and RF2 from the ribosome after translation termination[Freistroffer (1997) EMBO J 16 4126-4133]. The efficient recycling ofRF1 and RF2 by RF3-GTP, although dispensable for viability inprokaryotes, is required for maximum growth rate [Kisselev (2000) TrendsBiochem Sci 25, pp. 561-566].

In strains 26A6 and 33B7 the transposon has inserted in genes withhomology to the trmH family of tRNA/rRNA methyltransferases. Theinterrupted ORF in strain 26A3 shows greatest homology to H. influenzaeyibK, while that in strain 33B7 shows most similarity to yjfH of H.influenzae.

(iii) Regulation

Virulence genes involved in environmental sensing and coordinateregulation were identified in this screen. The A. pleuropneumoniaehomologue of the ferric uptake regulator, fur, was found to beinactivated in strain 6C12. Fur or Fur-like proteins have been shown toregulate virulence factor expression in a variety of pathogens includingE. coli(Shiga-like toxin), Corynebacterium diptheriae (diptheria toxin),and Pseudomonas aeruginosa (exotoxin A) [Prince (1991) Mol. Microbiol 5pp. 2823-2831; Schmitt (1991) Infect Immun 59, pp. 1899-1904; Calderwood(1987) J. Bcateriol 169, pp. 4759-4764]. Strains 21D3 and 19B10 containtransposon insertions in the A. pleuropneumoniae rpoE and upstream mclA(rseA) gene homologues, respectively. The rpoE encoded sigma factor,σ^(E)/Sigma-24, is a member of the extracytoplasmic function subfamilyof sigma factors that function as effector molecules in response toextracytoplasmic stimuli including heat shock and oxidative stress[Raina (1995) EMBO J 14, pp. 1043-1055; Rouviere (1995) EMBO J. 141032-1042; Ades (1999) Genes Dev 13, pp. 2449-2461; Dartigalongue (2001)J Biol Chem 276, pp. 20866-20875]. In S. typhimurium, σ ^(E) is animportant determinant of virulence and immunogenicity [Humphreys (1999)Infect Immun 67, pp. 1560-1568]. MclA (RseA) is an inner membraneprotein that acts as a σ^(E)-specific anti-σ factor. MclA (RseA) israpidly degraded in response to extracytoplasmic stress resulting inincreased σ^(E) concentration and initiation of the stress response[Ades (1999) Genes Dev 13, pp. 2449-2461]. Finally, insertions in the A.pleuropneumoniae quorum sensing luxS homologue (strain 26D3) wereobserved to attenuate A. pleuropneumoniae virulence. Recently, the E.coli luxS homologue has been implicated in the control of intestinalcolonisation factors encoded by the locus of enterocye effacementpathogenicity island in both EHEC and EPEC [Sperandio (2001) J Bacteriol183, pp. 5187-5197; Sperandio (1999) Proc Natl Acad Sci USA 96, pp.15196-15201].

(iv) Transport

The A. pleuropneumoniae gene aopA is inactivated in strain 23C9. TheaopA gene encodes a 48 kDa immunogenic outer membrane protein that iscommon to all serotypes of A. pleuropneumoniae tested [Cruz (1996)Infect Immun 64 pp. 83-90]. Interestingly, AopA is highly homologous(75% identity, 84% similarity) to Nqr1 of H. influenzae (NqrA of Vibrioalginolyticus), encoding the alpha chain of the sodium-translocatingNADH-ubiquinone oxidoreductase (NQR) complex [Hayashi (1996) FEBS Left381 pp. 174-176; Beattie (1994) FEBS Left 356 pp. 333-338]. The NQRenzyme is a respiration-linked Na⁺ pump establishing an electrochemicalgradient of sodium ions across the membrane. The resultant sodium motiveforce can be used for solute transport, ATP synthesis, and flagellarrotation. This alternative energy coupling of sodium ions rather thanprotons enables the bacteria to maintain a cytoplasmic pH nearneutrality in an alkaline environment. The NQR enzyme has been mostextensively studied in V. alginolyticus where it is thought to beinvolved in pH and ion homeostasis [Beattie (1994) FEBS Lett 356 pp.333-338; Unemoto (1993) J. Bioenerg Biomembr 25 pp. 385-391]. In Vibriocholerae, the NQR complex has been implicated in the regulation ofvirulence factor expression via its effects on oxT expression [Hase(1999) Proc Natl Acad Sci USA 96 pp. 3183-3187].

Components of several different putative ABC transport systems wereidentified as necessary for virulence of A. pleuropneumoniae. ABCtransport systems are involved in the transport (uptake or efflux) of adiverse array of macromolecules across the cytoplasmic membrane ofbacteria and eukaryotes. These systems usually consist of three basicparts: one or two ATPases, one or two integral membrane proteins and asubstrate-specific binding protein. Strains 13B12, 0C5, 19D1, and 24A4contain transposon insertions in the ATPase component of ABCtransporters. The gene inactivated in strain 0C5 encodes a protein withsignificant homology to MglA of prokaryotic galactose transporters(Richarme et al., J Biol Chem 268: 9473-9437, 1993). The in vivo CI ofthis mutant indicates limited availability of certain sugars within therespiratory tract environment. The interrupted ORFs in strains 19D1 and24A4 are homologous to hypothetical ATPases from transport systems ofunknown specificity in P. multocida (PM1728 and PM0996, respectively).In strain 20D10, the transposon has inactivated a gene with extensivehomology to znuA of H. ducreyi and pzpI of H. influenzae (Lewis et al.,Infect Immun 67: 5060-5068, 1999; Lu et al., J Biol Chem 272:29033-29038 1997 and FEMS Microbiol Lett 165:129-137 1998). ZnuA (PZP1)is a periplasmic zinc-binding protein that plays a key role in zincuptake. Inactivation of the znuA gene in H. ducreyi caused a significantdecrease in virulence in the rabbit model for experimental chancroid(Lewis et al., Infect Immun 67: 5060-5068, 1999). In strain 0D6 thetransposon has inserted in a gene encoding a protein with homology toputative integral membrane proteins from P. multocida (PM0997) and N.meningitidis (NMB0549). In P. multocida PM0997 is located immediatelyupstream from the ATPase inactivated in strain 24A4 suggesting that theyare part of the same transport system (May et al., Proc Natl Acad SciUSA 98: 3460-3465 2001). This gene was also identified by Fuller et al.(Fuller et al., Microb Pathog 29: 39-51 2000) as apvD, the predictedproduct of which shows homology with the macrolide-specific ABC-typeefflux protein MacA (Kobayashi et al., J Bacteriol 183: 5639-5644 2001).In mutant 9D10, the disrupted gene sequence encodes a protein withhomology to C or C which mediates both influx and efflux of magnesium aswell as cobalt efflux (Gibson et al., Mol Microbiol., 5: pgs 2753-2762,[1991]).

(v) Stress

In strain 13A3, the transposon has inserted in a dnaJ gene homologue.DnaJ is an essential component of bacterial Hsp70 chaperone systemswhere it functions as a co-chaperone, providing substrate specificity toits Hsp70 partner, DnaK [Rudiger (2001) EMBO J 20 pp. 1042-1050]. Inaddition to their fundamental roles in protein folding andtranslocation, DnaK/DnaJ constitute the primary stress-sensing andtransduction system of the E. coli heat shock response [Tomoyasu (1998)Mol Microbiol 30 pp. 567-581] where they modulate the induction of theheat shock response by altering the stability of the heat shock sigmafactor σ³². Significantly, mutations in DnaK have previously been shownto attenuate A. pleuropneumoniae virulence and to decrease the survivalof Brucella suis in human macrophage-like cell lines [Fuller (2000)Microb Pathog 29 pp. 39-51; Kohler (1996) Mol Microbiol 20 pp. 701-712].

Strain 26B6 contains an insertion in a htpG gene homologue. HtpG is aheat shock-induced molecular chaperone that, in E. coli, contributes tothe correct folding of cytoplasmic proteins in mildly stressed cells[Thomas (2000) Mol Microbiol 36 1360-1370]. HtpG does not appear toassociate stably with partner proteins and little is known about itsmechanism of action. It has been suggested that HtpG serves as a holderchaperone, transiently maintaining a subset of de novo synthesisedproteins in a conformation that is accessible to DnaK/DnaJ [Freeman(1996) EMBO J 15 pp. 2969-2979].

Strain 13D8 contains a transposon insertion in a Ion gene homologue. Lon(also called protease La) is a heat shock-inducible, ATP-dependentserine protease that plays a major role in the elimination of abnormallyfolded proteins [Gottesman (1996) Annu Rev Genet 30 465-506; Suzuki(1997) Trend Biochem Sci 22 pp. 118-123]. In addition to itsdemonstrated role as a stress response protease, Lon also functions as apleiotropic regulator of gene expression by degrading unstableregulatory proteins including RcsA, a positive regulator of capsulesynthesis [Keenleyside (1992) J Bacteriol 174 pp. 8-16] and SulA, aninhibitor of cell division and component of the E. coli SOS response[Mizusawa (1983) Proc Natl Acad Sci USA 80 pp. 358-362]. Recent studiesin Brjcella abortus have demonstrated that Lon is required for survivalin murine macrophages and wild-type virulence in the mouse model ofinfection [Robertson (2000) Mol Microbiol 35: 577-588].

Strain 13C1 carries a transposon insertion in a prc gene homologue. Prcis a periplasmic protease whose natural substrates appear to be membraneproteins including TonB [Gottesman (1996) Annu Rev Genet 30 pp.465-506]. It has been speculated that Prc functions as a chaperoneinvolved in the folding and turnover of proteins in the periplasmicspace [Bass (1996) J Bacteriol 178 pp. 1154-1161]. TABLE 1 Similarity toIn vitro In vivo Class Strain Gene % Span Number Known or PutativeFunction: CI: CI: Cell 9C2 cpxC AAD30161 capsular polysaccharide export1.020 3.88E−03 Surface inner membrane 26A9 IcbB (Nm) 37(50) 82 AAF21951LPS or capsule biosynthesis 1.068 9B7 galU AAC28326 LPS corebiosynthesis 1.750 25B7 rfbU AAG45944 LPS O-antigen biosynthesis 0.001410B11 mlC rfbC (Aa) 100 15 AAG49406 LPS O-antigen biosynthesis 0.23212D5 rfbN AAG45942 LPS O-antigen biosynthesis 0.024 21B8 rfbP AAG45943LPS O-antigen biosynthesis 0.029 15A9 hypothetical PF0798 (Pf) 37(55)270 gi: 18977170 LPS O-antigen biosynthesis 0.739 glycosyl- transferase4D4 IppB (Hs) 46(63) 83 AAK03698 lipoprotein B precursor 1.870 17A4ompP2 (Hi) 40(55) 237 Q48221 Outer membrane protein P2 1.149 precursorMetabolism 10B12 adpP (Hi) 61(76) 62 P44684 ADP-ribose pyrophosphatase1.029 10A11 argG (Hi) 65(88) 34 AAC23373 arginosuccinate synthase 0.80233C7 atpA (Hi) 89(95) 134 P43714 ATP synthase 0.314 17B8 ccmH (Hi)53(77) 166 P46458 cytochrome C biogenesis 1.100 protein 35D11 dsbA (Hi)63(77) 160 P31810 thiol: disulphide interchange 1.040 3.43E−04 proteinprecursor 26C3 hemA (Pm) 61(74) 394 CAA71452 Glutamyl-tRNA reductase0.048 4B9 moaA (Hi) 67(74) 114 P45311 molybdenum cofactor 1.150biosynthesis protein 19D5 mrp AAD01696 ATPase 0.200 29B11 napB (Pm)55(70) 77 AAK03681 periplasmic nitrate reductase 1.050 1.093    9B5 pnp(Pm) 75(79) 125 AAF68421 polynucleotide phosphorylase 1.180 OA7 pntB(Hi) 68(77) 458 P43010 NAD(P) transhydrogenase 0.785 23C9 aopA/nqrAAAC43631 Na-translocating NADH- 0.716 ubiquinone oxidoreductase 29A10prfC (Hi) 73(81) 342 P43928 peptide chain release factor 4.670 29B12recR (Hi) 81(87) 71 P44712 Recombination protein 3.600 26A10 thrC (Hi)88(93) 190 P44503 Threonine synthase 0.867 1.598    0F6 tonB (Hd) 64(74)288 O51810 Energy transducer 2.488 2.20E−02 27A12 tonB Y17916 Energytransducer 0.902 1.54    26D5 uroD/hemE (Pm) 88(93) 230 AAK03818Uroporphrinogen decarboxylase NA* 15A11 visC (Pm) 55(82) 43 AAK03810monooxygenase 0.684 26A6 yibK (Hi) 70(84) 127 P44868 probable tRNA/rRNA0.599 methyltransferase 33B7 yjfH (Hi) 77(85) 157 P44906 probable rRNAmethylase 0.744 26D12 guaA (Hi) 83(85) 43 P44335 GMP synthase 1.586Regulatory 6C12 fur (Hd) 79(83) 139 P71333 ferric uptake regulator 0.6272.76E−02 26D3 luxS (Hi) 69(84) 118 P44007 autoinducer-2 production 2.063protein 19B10 mlcA (Pm) 45(69) 61 AAK03872 negative regulator of rpoE1.725 6.50E−02 Stress 21D3 rpoE (Hi) 81(90) 189 P44790 Sigma factor E0.789 0.88    13A3 dnaJ (Hd) 85(99) 14 P48208 chaperone protein 0.16326B6 htpG (Ec) 69(82) 368 P10413 Heatshock protein 1.251 13D8 Ion (Pm)74(86) 292 AAK04062 ATP-dependent protease 0.674 13C1 prc (Pm) 65(79)290 AAK02353 tail specific protease 1.324 22A10 tig (Pm) 50(61) 152AAK04059 Trigger factor involved in cell 0.851 division Transport 9D10corC (Hi) 66(78) 248 Q57368 Magnesium and cobolt efflux 3.380 protein19D1 Hypothetical ABC 79(89) 110 AAK03812 ABC transporter 1.341 (Pm) 0C5mglA (Pm) 81(86) 242 P44884 galactoside ATP transporter 0.651 1.80E−020D6 Hypothetical 56(76) 210 AAK03081 Probable membrane protein 1.268membrane protein (Pm) 32A7 Hypothetical ABC 60(73) 370 AAK03080 ProbableABC transporter 0.962 (Pm) 13B12 yfeB (Pm) 75(86) 185 AAK02483 iron(III) ATP-binding protein 0.922 35D1 znuA (Hd) 71(78) 176 AAF00116periplasmic zinc ABC transporter 0.450 Unknown 14D5 No homology AUnknown 0.953 5.34E−03 2D5 No homology B Unknown 1.245 1.03E−02 32A11Unknown 31(52) 124 AAK03268 Unknown 2.950 0.991    (Pm) 4C1 Unknown42(59) 327 AAK02354 Unknown 1.110 0.935    (Pm) 9A4 Hypothetical 53(74)91 P44027 C4-decarboxylate transport 1.018 protein protein homologue(Hi)

1-40. (canceled)
 41. An attenuated Actinobacillus pleuropneumoniaebacterium.
 42. The bacterium of claim 41, having a mutation in a generequired for bacterial virulence.
 43. The bacterium of claim 41, havinga mutation in a gene which comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NO.:1-56.
 44. The bacterium of claim 41,having a plurality of mutations, occurring within a single gene orwithin different genes.
 45. A composition, comprising the bacterium ofclaim
 41. 46. The composition of claim 45, comprising a plurality ofdifferent attenuated A. pleuropneumoniae bacteria selected from thegroup consisting of bacteria having different mutations in the samevirulence gene, bacteria having similar or different mutations in two ormore different genes, and mixtures thereof.
 47. A method of treating anorganism, comprising: administering to the organism the bacterium ofclaim 41; where the treatment is selected from the group consisting ofpreventing an infection with A. pleuropneumoniae, alleviating aninfection with A. pleuropneumoniae, preventing symptoms associated withA. pleuropneumoniae infection, and alleviating symptoms associated withA. pleuropneumoniae infection.
 48. An attenuated bacterium having amutation in a gene comprising a nucleotide sequence which is capable ofhybridising to any one of the nucleotide sequences defined by SEQ IDNO:1-56, under conditions of moderate to high stringency.
 49. A methodof treating an organism, comprising: administering to the organism theattenuated bacterium of claim 48; where the treatment is selected fromthe group consisting of preventing an infection with a wild-typebacterium (or a different strain or serotype thereof, alleviating aninfection with a wild-type bacterium (or a different strain or serotypethereof, preventing symptoms associated with an infection with awild-type bacterium (or a different strain or serotype thereof, andalleviating symptoms associated with an infection with a wild-typebacterium (or a different strain or serotype thereof.
 50. An isolatedpolynucleotide, selected from the group consisting of: a polynucleotideencoding a gene product which is naturally involved in (e.g. requiredfor) the virulence of A. pleuropneumoniae; a polynucleotide encoding agene product which is not naturally found in A. pleuropneumoniae, butwhose expression therein is capable of modulating (e.g. of decreasing)the virulence of that bacterium; a polynucleotide which is not naturallyfound in A. pleuropneumoniae but which is capable of modulating thevirulence of that bacterium by its direct interaction with A.pleuropneumoniae virulence genes or gene products; and a polynucleotidecomprising (a) a nucleotide sequence selected from the group consistingof SEQ ID NO.: 1-56; (b) a nucleotide sequence encoding the polypeptidewhich is encoded by the nucleotide sequence recited in (a); (c) anucleotide sequence which hybridizes to the nucleotide sequence of (a)and/or (b), or to its complement, under conditions of moderate to highstringency; or (d) a fragment of any one of the nucleotide sequences of(a)-(c), which fragment retains an immunological property and/or abiological activity of the recited nucleotide sequence of (a)-(c).
 51. Avector comprising the polynucleotide of claim
 50. 52. A host cellcontaining the polynucleotide of claim
 50. 53. An isolated A.pleuropneumoniae virulence polypeptide.
 54. A virulence polypeptideencoded by the polynucleotide of claim
 50. 55. A method of producing avirulence polypeptide, comprising: (i) culturing the host cell of claim52 under conditions that permit the expression of the polypeptide; and(ii) recovering and optionally isolating the expressed polypeptide fromthe host cell, or from its surrounding medium.
 56. A compositioncomprising an isolated A. pleuropneumoniae virulence polypeptide or thepolypeptide of claim
 54. 57. An antibody which specifically recognizesthe polynucleotide of claim 50, a polypeptide encoded by thepolynucleotide, or an isolated A. pleuropneumoniae polypeptide.
 58. Amethod for identifying an anti-bacterial agent which is capable ofmodulating the function of an A. pleuropneumoniae virulence gene, or ofa homologous gene in a related species, comprising: screening potentialagents for their ability to interfere with the expression and/orbiological activity in a host bacterium of the gene products encoded bythe nucleotide sequences set forth in any one of SEQ ID NO:.1-56.
 59. Ananti-bacterial agent identified by the method of claim
 58. 60. A methodof treating an animal suffering from a Pasteurellaceae (e.g. an A.pleuropneumoniae) infection, comprising: administering theanti-bacterial agent of claim 59.